The estrogens are essential hormones for regulating normal development, growth and reproductive functions. The estrogens, as well as the estrogenic antagonists mediate their biological actions by their interactions with the estrogen receptor. The estrogen receptor in turn regulates the transcriptional activity of estrogen responsive genes by its interactions with specific DNA sequence of these genes referred to as the estrogen responsive elements (ERE). The long term goal of this research program is to elucidate the mechanism of estrogen and estrogenic antagonist interactions with the estrogen receptor, and to gain insight into the structure-function relationship of the receptor. The estrogen receptor is phosphorylated in an estrogen dependent manner immediately before or after binding to an ERE and as a consequence the gene's transcriptional activity is promoted. A major gap in our knowledge and the focus of this research program is to describe the mechanism(s) by which the estrogen dependent phosphorylation of the estrogen receptor regulates the receptor's transcriptional function. Our working model is that as a consequence of phosphorylation of the estrogen receptor the receptor affinity for its ERE is increased and/or changes the conformation of the estrogen receptor-ERE complexes in such a way to promote transcriptional activity. We will identify which crucial serine(s) of the estrogen receptor is (are) phosphorylated in an estrogen dependent manner We intend to characterize and identify the specific protein kinase responsible for the phosphorylation of the receptor. The in vivo transcriptional activity of the crucial serine(s) that is phosphorylated will be assessed using site-directed mutagenesis of the specific serine(s). We will determine whether phosphorylation of the estrogen receptor influences its interaction with the ERE to antagonists to promote transcriptional activation. The ability of estrogens and estrogenic antagonists to phosphorylate the crucial serine(s) will be determined by [32p] labeling of the estrogen receptor in MCF-7 cells followed by receptor purification and SDS-gel electrophoresis. HPLC isolation of the [32p]labeled tryptic peptide(s) and gas-phase microsequencing of the peptide(s) will be used to identify the crucial serine(s) that is phosphorylated in a hormone dependent manner. These studies will provide a test of our model and an important insight into a key step by which the estrogen receptor regulates gene transcription.